Regulatory T cells suppress autoimmunity

ABSTRACT

The invention provides methods for producing an autoantigen-specific regulatory T Cell enriched composition, and resultant compositions and methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Pat Appl Ser No. 60/535,085,filed Jan. 8, 2004.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This work was supported by NIH NCRR Grant R37 A146643. The U.S.government may have rights in any patent issuing on this application.

INTRODUCTION

Background of the Invention

Autoimmune diseases comprise many of the most devastating andintractable ailments today, where representative autoimmune diseasesinclude diabetes mellitus, uveoretinitis and multiple sclerosis, amongothers.

The potential for Tregs to actively regulate autoimmunity and inducelong term tolerance has great potential application as a strategy forinducing long-lived tolerance. Taking advantage of Tregs has beencomplicated by an inability to expand and characterize this minor T cellsubset, a population of cells reduced even further in autoimmune-proneanimals and patients. For instance, recent studies have suggested thatit may be impossible to reverse ongoing autoimmune diabetes due to theautoreactive T cells becoming resistant to suppression during the activephase of the disease. Prior efforts to expand Tregs ex vivo have notachieved clinically sufficient expansion, nor demonstrable in vivoefficacy (e.g. Fu et al., 2004, Am J Transplant. 4, 65-78). The lownumber of CD4+ CD25+regulatory T cells (Tregs), their anergic phenotypeand diverse antigen specificity present major challenges to harnessingthis potent tolerogenic population to treat autoimmunity and transplantrejection.

A number of US patent documents relate to T cell expansion, includingHorwitz (e.g. U.S. Pat. Nos. 6,803,036 and 6,797,267, and related patentpublications); U.S. Pat. No. 6,534,055; U.S. 2003/124122A1; U.S.2003/0082806A1; U.S. 2002/0058019A1; U.S. 2002/0119568A1; U.S.2003/0119185A1; and U.S. 2002/0019048A1.

SUMMARY OF THE INVENTION

The invention provides methods for producing an autoantigen-specificregulatory T cell enriched composition, and resultant compositions andmethods of use. In one embodiment, the invention provides a method ofmodulating an autoimmune reaction in a subject, said method comprising(a) obtaining a population of subject-compatible cells; (b) producing anautoantigen-specific regulatory T cell enriched composition from saidpopulation of cells; and (c) introducing said composition into saidsubject to modulate said autoimmune reaction in said subject.

In particular embodiments, the population of cells is obtained from saidsubject, obtained from a donor distinct from said subject, and/orharvested from peripheral blood.

In particular embodiments, the producing step comprises expanding saidantigen-specific regulatory T cells, and/or enriching saidautoantigen-specific regulatory T cells from said obtained population ofcells.

In particular embodiments, the expanding is achieved by contacting saidpopulation of cells with an autoantigen-specific regulatory T cellstimulatory composition.

In particular embodiments, the regulatory T cells are enriched from saidpopulation of cells prior to said expanding step, or after saidexpanding step.

In particular embodiments, the stimulatory composition comprises an MHCclass II/autoantigenic peptide complex, a costimulatory agent or asecond regulatory T cell stimulatory agent.

In particular embodiments, the costimulatory agent is an agonistantibody, such as an agonist antibody which binds to CD28.

In particular embodiments, the second stimulating agent is a cytokine,such as an interleukin, such as interleukin-2.

In particular embodiments, the stimulatory composition is immobilized ona substrate, such as a cell or bead.

In particular embodiments, the producing step comprises

In particular embodiments, the said modulating comprises inhibiting.

The invention also provides compositions comprising a population ofcells wherein at least 50% of said cells of said composition are naturalautoantigen-specific regulatory T cells.

In particular embodiments, the autoantigen-specific regulatory T cellsare specific for peptides presented in MHC class II molecules as shownin Table A.

In particular embodiments, the autoantigen-specific regulatory T cellsare effective at modulating an autoimmune reaction when administered toa subject.

The invention also provides kits for producing a composition ofautoantigen-specific regulatory T cells, said kit comprising: (a) anautoantigen-specific T cell receptor stimulatory agent; and (b) acostimulatory agent.

In particular embodiments, the stimulatory agent is an MHC classII/autoantigenic peptide complex.

In particular embodiments, the costimulatory agent is an agonistantibody, such as an antibody which binds to CD28.

In particular embodiments, the kit further comprises a second regulatoryT cell stimulating agent, such as a cytokine, such as an interleukin,such as interleukin-2 or interleukin-15.

In particular embodiments, the stimulatory agent and said costimulatoryagent are immobilized on a substrate, such as a cell or bead.

The invention provides methods and compositions for ex vivo expansion oftherapeutic regulatory T cells, and resultant compositions and methodsof use. The expansion methods generally comprise the steps of: isolatingfrom a mixed population of T cells a subpopulation enriched inCD4⁺CD25⁺T cells (Treg cells); expanding the Treg cells of thesubpopulation by contacting the subpopulation with effective amounts of(i) a TCR/CD3 activator (ii) a TCR costimulator activator and (iii)IL-2, to obtain ex vivo expanded Treg cells, wherein the expanded Tregcells demonstrate immune suppression, wherein the isolating step istypically prefaced by extracting the population from a person orpatient, typically suffering or in remission from an autoimmune diseaseamenable to therapy as described herein.

In particular embodiments, the subpopulation comprises >98% Treg cells,preferably >98% CD4⁺CD25⁺CD62L⁺ Treg cells; the isolation step comprisesnegative and positive immuno-selection and cell sorting; the expandingstep effects at least a 100-fold expansion of the subpopulation; theTCR/CD3 activator is a multivalent antibody or ligand for TCR/CD3; theTCR costimulator activator is a multivalent antibody or ligand for CD28,GITR, B7-1/2, CD5, ICOS, OX40 or CD40; the effective amount of IL-2 is200 to 2500 IU IL-2/ml; and/or the Treg cells suppress proliferation ofanti-CD3 or alloantigen stimulated CD25⁻ T cells in vitro, orautoimmunity, including graft-versus-host disease in vivo.

In more particular embodiments:

-   -   an effective amount of the ex vivo expanded Treg cells        introduced into the patient diagnosed with diabetes mellitus and        presenting an indication of impaired glucose homoeostasis        selected from fasting plasma glucose (FPG), post-prandial        glucose (PPG), and glucose tolerance (GTT) provide a resultant        improvement in the impaired glucose homoeostasis, wherein the        improvement is preferably selected from an FPG of 110 mg/dL or        less, a 2-hour PPG of 140 mg/dL or less, and a GTT of 140 mg/dL        or less 2 hours after a 75-g glucose load;    -   the TCR/CD3 activator is an anti-CD3 antibody, and the TCR        costimulator activator is an anti-CD28 antibody, wherein the        anti-CD3 and anti-CD28 antibodies are immobilized on        paramagnetic beads provided in a Treg cell:bead ratio of between        1:1 and 1:2;    -   the TCR/CD3 activator and the expanded Treg cells are        antigen-specific, preferably wherein the TCR/CD3 activator is an        MHC-peptide multimer, wherein the peptide is a        diabetes-associated autoantigen peptide and the        diabetes-associated autoantigen is selected from glutamic acid        decarboxylase (GAD), an islet cell autoantigen (ICA) and        insulin, and the TCR costimulator activator is an anti-CD28        antibody.

The invention also provides methods and compositions for adoptivecellular immunotherapy comprising the step of introducing into a patientin need thereof an effective amount of the subject ex vivo expanded Tregcells. These methods generally comprise the steps of: extracting a mixedpopulation of T cells from a person; isolating from the population asubpopulation enriched in CD4⁺CD25⁺T cells (Treg cells); expanding theTreg cells of the subpopulation by contacting the subpopulation witheffective amounts of (i) a TCR/CD3 activator, (ii) a TCR costimulatoractivator, and (iii) IL-2, to obtain ex vivo expanded Treg cells;introducing into a patient an effective amount of the ex vivo expandedTreg cells; and detecting a resultant suppression of autoimmunity.

In particular embodiments, the person and patient is a patient diagnosedwith diabetes mellitus and presenting an indication of impaired glucosehomoeostasis selected from fasting plasma glucose (FPG), post-prandialglucose (PPG), and glucose tolerance (GTT); the subpopulationcomprises >98% Treg cells; the subpopulation comprises >98%CD4⁺CD25⁺CD62L+Treg cells; the isolation step comprises negative andpositive immuno-selection and cell sorting; the expanding step effectsat least a 100-fold expansion of the subpopulation; the TCR/CD3activator is selected from a multivalent antibody or ligand for TCR/CD3;the TCR costimulator activator is a multivalent antibody or ligand forCD28, GITR, CD5, ICOS, OX40 or CD40L; the effective amount of IL-2 is200 to 2500 IU IL-2/ml; the Treg cells suppress proliferation ofanti-CD3 or alloantigen stimulated CD25⁻ T cells, and/or the resultantsuppression of autoimmunity is detected as a resultant improvement inthe impaired glucose homoeostasis.

In more particular embodiments:

-   -   the improvement is selected from an FPG of 110 mg/dL or less, a        2-hour PPG of 140 mg/dL or less, and a GTT of 140 mg/dL or less        2 hours after a 75-g glucose load;    -   the TCR/CD3 activator is an anti-CD3 antibody, and the TCR        costimulator activator is an anti-CD28 antibody, wherein the        anti-CD3 and anti-CD28 antibodies are immobilized on        paramagnetic beads provided in a Treg cell:bead ratio of between        1:1 and 1:2; and/or the TCR/CD3 activator is an MHC-peptide        multimer, wherein the peptide is a diabetes-associated        autoantigen peptide and the diabetes-associated autoantigen is        selected from glutamic acid decarboxylase (GAD), an islet cell        autoantigen (ICA) and insulin, and the TCR costimulator        activator is an anti-CD28 antibody.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The invention provides methods for producing a predeterminedautoantigen-specific regulatory T cell enriched composition, andresultant compositions and methods of use. In one embodiment, theinvention provides a method of modulating an autoimmune reaction in asubject, said method comprising (a) obtaining a population ofsubject-compatible cells; (b) producing an autoantigen-specific,preferably predetermined autoantigen-specific regulatory T cell enrichedcomposition from said population of cells; and (c) introducing saidcomposition into said subject to modulate said autoimmune reaction insaid subject.

In particular embodiments, the population of cells is obtained from saidsubject, obtained from a donor distinct from said subject, and/orharvested from peripheral blood. The population of cells obtainedcomprises autoantigen-specific regulatory T (Treg) cells, and may bederived from any source in which autoantigen-specific Treg cells exist,such as peripheral blood, the thymus, lymph nodes, spleen, and bonemarrow. In certain embodiments, the source of Treg cells may be fromcadaveric tissue.

The population of cells may be obtained from the subject into which theTreg-enriched composition is subsequently introduced. The subject can beany mammal in which modulation of an autoimmune reaction is desired.Mammals of interest include, but are not limited to: rodents, e.g. mice,rats; livestock, e.g. pigs, horses, cows, etc., pets, e.g. dogs, cats;and primates, e.g. humans. In one embodiment, the subject is an animalmodel of an autoimmune disease. There are numerous, established animalmodels for using T cell epitopes of autoantigens to induce tolerance,including multiple sclerosis (EAE: experimental autoimmuneencephalomyelitis), myasthenia gravis (EMG: experimental myastheniagravis) and neuritis (EAN: experimental autoimmune neuritis). In anotherembodiment, the subject is a human afflicted with an autoimmune diseaseor disorder, such as any of the diseases/disorders listed in Table A.

In an alternate embodiment, the population of cells is obtained from adonor distinct from the subject. The donor is preferably syngeneic, butcan also be allogeneic, or even xenogeneic provided the cells obtainedare subject-compatible in that they can be introduced into the subject,optionally in conjunction with an immunosuppressive therapy, withoutresulting in extensive chronic graft versus host disease (GvHD).Allogeneic donor cells are preferably human-leukocyte-antigen(HLA)-compatible, and are typically administered in conjunction withimmunosuppressive therapy. To be rendered subject-compatible, xenogeniccells may be subject to gamma irradiation or PEN1 0 treatment (Fast, L Det al, Transfusion. 2004 February; 44(2):282-5).

The producing step provides a predetermined autoantigen-specificregulatory T cell enriched composition from said population of cells,preferably specific for a predetermined autoantigen associated with thetargeted autoimmune reaction, preferably predetermined to be associatedwith the targeted autoimmune reaction. In particular embodiments, theproducing step comprises expanding said antigen-specific regulatory Tcells, and/or enriching said autoantigen-specific regulatory T cellsfrom said obtained population of cells.

An autoantigen-specific regulatory T (Treg) cell enriched composition isone in which the percentage of autoantigen-specific Treg cells is higherthan the percentage of autoantigen-specific Treg cells in the originallyobtained population of cells. In particular embodiments, at least 75%,85%, 90%, 95%, or 98% of said cells of the composition areautoantigen-specific regulatory T cells. In particular embodiments, theproducing step comprises expanding the antigen-specific regulatory Tcells, and/or enriching said autoantigen-specific regulatory T cellsfrom said obtained population of cells.

In particular embodiments, the regulatory T cells are enriched from saidpopulation of cells prior to said expanding step, or after saidexpanding step. Treg cells can be enriched by targeting for selection ofcell surface markers specific for immune suppressive Tregs andseparating using automated cell sorting such as fluorescence-activatedcell sorting (FACS), solid-phase magnetic beads, etc, as described belowin Examples 1 and 2. To enhance enrichment, positive selection may becombined with negative selection against cells comprising surface makersspecific to non-Treg cell types, such as depletion of CD8, CD11b, CD16,CD19, CD36 and CD56-bearing cells, and as exemplified below.

In particular embodiments, the expanding is achieved by contacting thepopulation of cells with an autoantigen-specific regulatory T cellstimulatory composition. The autoantigen-specific regulatory T cells arepreferably expanded at least 50-fold, and preferably at least 100, 200,300, 500 and 800-fold. Autoantigen-specific regulatory T cellstimulatory compositions promote the survival, growth, and/or expansionof autoantigen-specific regulatory T cells that express T cellreceptor(s) that recognize a desired autoantigen.

Preferred stimulatory compositions stimulate the T cell byantigen-specifically binding and activating the T cell receptor complex.A variety of antigen-specific TCR-binding reagents may be used,including cross-linked peptide-bound MHC molecules, antibodies, andmimetics. In a preferred embodiment, the compositions comprises an MHCclass I/autoantigenic peptide complex, particularly an aggregate of suchMHC/peptide complexes. These complexes comprises at least theextracellular peptide binding domain of an MHC class II molecule inwhich is functionally bound an autoantigenic peptide. The complexes canbe in solution or suspension or immobilized on a substrate, such aspresented on the surface of a cell, particularly an APC. Numerousapplicable methods are known in the art for generating functional MHCclass I/peptide complexes, such as may be found in literu In oneembodiment, the autoantigenic peptide is a peptide of the naturallyoccurring autoantigen that is capable of complexing with an MHC class IImolecule. Exemplary MHC class II molecules/peptide complexes are listedin Table A. In an alternative embodiment, the autoantigenic peptide is amimotope peptide capable of complexing with an MHC class II molecule.

In another embodiment the autoantigenic peptide is a mimotope peptidethat is capable of complexing with an MHC class II molecule. Mimotopepeptides are described in the literature, further below, and inExamples 1. Protocols for using autoantigen peptides to expand Tregsfrom otherwise conventional T cells include the use ofautoantigen-specific MHC-peptide tetramers, peptide-pulsed DCs(Yamazaki, et al, 2003, J Exp Med 198:235-47) or artificial APCs (Mauset al. Nat. Biotechnol. 20:143-8, 2002) to expand Tregs from patientsindependent of the cell surface phenotype. In addition, a combination ofin vitro and in vivo approaches can enhance the effects of the therapy.For example, recent studies have shown that administration of selfantigens, altered peptide ligands and even non-specific stimuli such asFcR non-binding anti-CD3 mAbs can promote antigen-specific Treg activity(Apostolou et al. J. Exp. Med. 199:1401-8, 2003; Belghith et al. Nat.Med. 9:1202-8, 2003). Hence, combining in vivo immunization to inducethe Tregs with ex vivo expansion or visa versa may be advantageous.

In certain embodiments, the stimulatory composition may further includeone or more additional agents, e.g., a costimulatory agent, a secondregulatory T cell stimulatory agent, or agents that generally promotethe survival and/or growth of T cells.

In certain embodiments, the costimulatory agent is an antibody or ligandspecific for a TCR costimulator, such as CD28 or GITR, as describedbelow. In particular embodiments, the costimulatory agent is an agonistantibody, such as an agonist antibody which binds to CD28.

The stimulatory composition alternatively comprises a second regulatoryT cell stimulatory agent. Exemplary stimulatory agents includegranulocyte colony stimulating factor, interleukins such as IL-2, IL-6,IL-7, IL-1 3, and IL-1 5, and hepatocyte growth factor (HGF). Inparticular embodiments, the second stimulating agent is a cytokine, suchas an interleukin, such as interleukin-2.

In particular embodiments, one or more components of the stimulatorycomposition is immobilized on a substrate, such as a cell or bead. Cellssuitable for use as substrates include artificial antigen-presentingcells (AAPCs) (Kim, J V et al, Nat Biotechnol. 2004 April; 22(4):403-10;and Thomas, A K et al, Clin Immunol. 2002 December; 105(3):259-72).Beads can be plastic, glass, or any other suitable material, typicallyin the 1-20 micron range. Paramagnetic beads are preferred.

Optimal concentrations of each component of the stimulatorycompositions, culture conditions and duration can be determinedempirically using routine experimentation. An exemplaryautoantigen-specific regulatory T cell stimulatory composition isdescribed in Example 2.

The expanded and/or enriched autoantigen-specific regulatory T cells areintroduced into the subject to modulate an autoimmune reaction. Forexample, the subject may be afflicted with a disease or disordercharacterized by having an ongoing or recurring autoimmune reaction,such as the diseases/disorders listed in Table A. In particularembodiments, the said modulating comprises inhibiting. Tregs may serveas a “Trojan Horse” to deliver suppressive or other biologic factors tosites of inflammation, such as IL-4 (Yamamoto et al. J. Immunol.166:4973-80, 2001), stem cell growth factors, angiogenesis regulators,genetic deficiencies, etc. For example, overexpression of foxp3 has beenshown to transform otherwise pathogenic T cells into Tregs (Jaeckel etal. Diabetes. 2004 Dec. 10; [Epub]), and polyclonally expanded Tregs canbe transduced with genes encoding an antigen-specific TCR plus foxp3 togenerate potent antigen-specific Tregs in very high numbers andefficiency (Mekala, et al., Blood. 2004 Nov. 4; [Epub]). Thus, theseantigen-specific approaches decrease the requirement for high cellnumbers while maximizing Treg specificity and function.

Antigen-specific Tregs are particularly indicated in infectious diseasesin which the pathogenicity of the infections is not a result of thecytopathic effects of the pathogen but rather the tissue damage causedby the immunoinflammatory response to the infectious agent. In diseases,such as hepatitis C or HSV-induced corneal inflammation, Treg therapyprovides a unique opportunity to control viral-inducedimmunoinflammatory disease (Suvas et al. J. Immunol. 172: 4123-4132,2004). Viruses, such as Coxsackie, are known to cause pancreatitis andhave been associated with the development of Type 1 Diabetes. Thus,Tregs that target expressed viral antigens can be used to suppress localtissue damage caused by the infection and reduce the inflammation thatincites autoimmune disease development.

The invention also provides compositions comprising a population ofcells wherein at least 50% of said cells of said composition are natural(nontransformed), preferably expanded autoantigen-specific regulatory Tcells, wherein the autoantigen-specificity is preferably predetermined,preferably predetermined to a targeted autoimmune reaction antigen. Thecompositions are made by the methods described herein. The percentage ofthe autoantigen-specific regulatory T cells in the composition can beascertained using the methodology described in Example. 2. In particularembodiments, at least 75%, 85%, 90%, 95%, or 98% of said cells of thecomposition are autoantigen-specific regulatory T cells.

In particular embodiments, the autoantigen-specific regulatory T cellsare specific for an MHC class II molecule/peptide complex listed inTable A.

In particular embodiments, the autoantigen-specific regulatory T cellsare effective at modulating an autoimmune reaction when administered toa subject. Effective and optimized dosages and treatment regimes usingthe expanded and/or enriched autoantigen-specific regulatory cells areinformed from vast clinical experience with existing T-cell infusiontherapies, and can be further determined empirically.

The subject methods find use in the treatment of a variety of differentconditions in which the modulation of an aberrant immune response in thehost is desired. By aberrant immune response in a host is meant anyimmune reaction in a subject characterized as an autoimmune response(e.g., an autoimmune disease). In general, autoimmune responses occurwhen the immune system of a subject recognizes self-antigens as foreign,leading to the production of self-reactive effector immune cells. Selfreactive effector immune cells include cells from a variety of lineages,including, but not limited to, cytotoxic T cells, helper T cells, and Bcells. While the precise mechanisms differ, the presence of autoreactiveeffector immune cells in a host suffering from an autoimmune diseaseleads to the destruction of tissues and cells of the host, resulting inpathologic symptoms. Numerous assays for determining the presence ofsuch cells in a host, and therefore the presence of an autoimmunedisease, such as an antigen specific autoimmune disease in a host, areknown to those of skill in the art and readily employed in the subjectmethods. Assays of interest include, but are not limited to, thosedescribed in: Autoimmunity. 2003 September-November; 36(6-7):361-6; JPediatr Hematol Oncol. 2003 December; 25 Suppl 1:S57-61; Proteomics.2003 November; 3(11):2077-84; Autoimmun Rev. 2003 January; 2(1):43-9.

By treatment is meant that at least an amelioration of the symptomsassociated with the aberrant immune response in the host is achieved,where amelioration is used in a broad sense to refer to at least areduction in the magnitude of a parameter, e.g. symptom, associated withthe condition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g. prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from thecondition, or at least the symptoms that characterize the condition.

A variety of hosts are treatable according to the subject methods. Incertain embodiments, such hosts are “mammals” or “mammalian,” wherethese terms are used broadly to describe organisms which are within theclass mammalia, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g.,humans, chimpanzees, and monkeys). In many embodiments, the hosts willbe humans.

In further embodiments, the methods include a step of diagnosing thepresence of an autoimmune disease. By diagnosing is meant that theautoimmune response of a subject is generally classified, e.g, diabetesmellitus, SLE, MS, etc. Further, at least one autoantigen is identifiedto which the aberrant immune response is directed. A variety ofdiagnostic methods are known in the art and are currently beingdeveloped. As such, the methods of the subject invention are not limitedto specific assays for diagnosing the autoimmune disease in a host orthe antigen to which it is directed.

Also provided are reagents and kits thereof for practicing one or moreof the above-described methods. The subject reagents and kits thereofmay vary greatly. In certain embodiments, the kits include at least anantigen specific regulatory T cell stimulatory composition. In otherembodiments, the kit includes another regulatory T cell stimulatingagent, such as a cytokine, such as an interleukin, such as interleukin-2or interleukin-15. In certain embodiments, the kits may further includereagents for performing the antigen specific regulatory T cell expansionstep, including culture dishes or flasks, culture medium, or anynecessary buffers, factors, etc. In yet other embodiments, the kitsinclude the means to harvest the sample containing the regulatory Tcells and the reagents necessary to perform regulatory T cellenrichment/purification.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

In particular embodiments, the stimulatory agent is an MHC classI/autoantigenic peptide complex. Exemplary MHC class IImolecules/peptide complexes are listed in Table A.

The costimulatory agent is an antibody or ligand specific for a TCRcostimulator, such as CD28 or GITR, as described below. In particularembodiments, the costimulatory agent is an agonist antibody, such as anantibody which binds to CD28.

In particular embodiments, the stimulatory agent and said costimulatoryagent are immobilized on a substrate, such as a cell or bead.

The invention provides methods and compositions for ex vivo expansion oftherapeutic regulatory T cells (Treg cells), and the use of suchexpanded Treg cells for adoptive cellular immunotherapy to suppressautoimmunity.

The expansion methods generally comprise first extracting a mixedpopulation of T cells from a person or patient, and isolating from thepopulation a subpopulation enriched in Treg cells. To maximize efficacy,the subpopulation is enriched to at least 90%, preferably at least 95%,and more preferably at least 98% Treg cells, preferably CD4⁺CD25⁺CD62L⁺Treg cells. Cells are generally enriched by targeting for selection cellsurface markers specific for immune suppressive Tregs and separatingusing automated cell sorting such as fluorescence-activated cell sorting(FACS), solid-phase magnetic beads, etc. To enhance enrichment, positiveselection may be combined with negative selection against cellscomprising surface makers specific to non-Treg cell types, such asdepletion of CD8, CD11b, CD16, CD19, CD36 and CD56-bearing cells, and asexemplified below.

The Treg-enriched subpopulation is then expanded ex vivo by culturingthe cells in the presence of effective amounts of a TCR/CD3 activator, aTCR costimulator activator, and IL-2. The TCR/CD3 activator is selectedfrom a multivalent antibody or ligand for TCR/CD3, including antigennon-specific activators such as an anti-CD3 antibody, andantigen-specific activators, such as an MHC-peptide multimer (see, e.g.Yee, et al., Adoptive T cell therapy using antigen-specific CD8+ T cellclones for the treatment of patients with metastatic melanoma: In vivopersistence, migration, and antitumor effect of transferred T cells.Proc Natl Acad Sci USA, Dec. 10, 2002; 99(25): 16168-16173; Butterfield,et al., T-Cell responses to HLA-A*0201 immunodominant peptides derivedfrom a-fetoprotein in patients with hepatocellular cancer, Clin. CancerRes., Dec. 1, 2003; 9(16): 5902-5908; and Yee, et al., Isolation of highavidity melanoma-reactive CTL from heterogeneous populations usingpeptide-MHC tetramers, J Immunol, 1999, 162: 2227-223), wherein thepeptide is typically an autoimmune disease associated peptide, such as adiabetes-associated autoantigen peptide wherein suitablediabetes-associated autoantigens include glutamic acid decarboxylase(GAD), an islet cell autoantigen (ICA) and insulin, wherein combinationsof such peptides may also be used.

The costimulator activator is a multivalent antibody or ligand specificfor a TCR costimulator, preferably CD28 or GITR (Shimizu et al.,Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaksimmunological self-tolerance, Nat Immunol. 2002 February; 3(2): 135-42.Epub 2002 Jan. 22; Tone et al., Mouse glucocorticoid-induced tumornecrosis factor receptor ligand is costimulatory for T cells, Proc NatlAcad Sci USA. 2003 Dec. 9; 100(25):15059-64. Epub 2003 Nov. 07), thoughalternative TCR costimulators such as CD5, ICOS, OX40 and CD40L may alsobe targeted where suitable expansion is so obtained, as may bedetermined empirically. To promote activation and expansion, the TCR/CD3and TCR costimulator activators are typically immobilized on a3-dimensional solid surface, such as a host cell (e.g. Thomas et al,December 2002, Clin Immunol 105, 259-72) or bead. In a particularembodiment, the activators are immobilized on paramagnetic beadsprovided in a Treg cell:bead ratio of between 2:1 and 1:5, preferablybetween 1:1 and 1:3. Optimal bead size is empirically determined, thoughtypically in the range of 1 to 20 micron diameters.

The IL-2 is typically presented in recombinant form, wherein effectiveamounts of IL-2 are typically 200 to 2500 IU IL-2/ml. We have foundincreased expansions using unconventionally elevated IL-2 concentrationsranging from 500-2500, and preferably 1000-2000 IU IL-2/ml. The targetTreg cells of the subpopulation are preferably expanded at least50-fold, and preferably at least 100, 200 or 300-fold. Maximalexpansions are determined empirically and will vary by cell type,incubation conditions, etc. For exemplified embodiments, maximalexpansions are found to be about 300, 500 and 800-fold.

The suppressive function of the expanded Treg cells may be detected invitro or in vivo. For example, in vitro, the expanded Treg cells may beshown to suppress proliferation of CD25⁻ T cells stimulated withanti-CD3 in the presence of Fc-receptor-bearing cells, or CD25⁻ T cellsstimulated with irradiated allogeneic splenocytes. Suitable exemplary invivo animal model and human clinical immune suppression protocols aredescribed further below.

In particular embodiments, the TCR/CD3 activator and the expanded Tregcells are autoantigen-specific. For example, in a particular suchembodiment, an effective amount of the ex vivo expanded Treg cellsintroduced into the patient diagnosed with diabetes mellitus (see, e.g.Mayfield et al., Diagnosis and classification of diabetes mellitus: newcriteria, Am Fam Physician. 1998 Oct. 15; 58(6):1355-62, 1369-70) andpresenting an indication of impaired glucose homoeostasis, such asfasting plasma glucose (FPG), post-prandial glucose (PPG), and glucosetolerance (GTT) provide a resultant improvement in the impaired glucosehomoeostasis, particularly wherein the improvement is selected from anFPG of 110 mg/dL or less, a 2-hour PPG of 140 mg/dL or less, and a GTTof 140 mg/dL or less 2 hours after a 75-g glucose load. Accordingly, theinvention provides methods and compositions for adoptive cellularimmunotherapy comprising introducing into a patient in need thereof aneffective amount of the subject ex vivo expanded Treg cells.

These applications generally involve reintroducing expanded Treg cellsextracted from the same patient, though the methods are also applicableto adoptive cellular immunotherapy for treatment of graft-versus-hostdisease associated with transplantation, particularly bone marrowtransplantation using Tregs derived from donor tissue.

Adoptive transfer of Tregs expanded as disclosed herein is effective tosuppress a wide variety of pathogenic autoimmune responses, includingdiabetes, GVHD, Lupus, rheumatoid arthritis, psoriasis, multiplesclerosis, degenerative heart disease (e.g. Ziad Mallat, et al.Induction of a Regulatory T Cell Type 1 Response Reduces the Developmentof Atherosclerosis in Apolipoprotein EBKnockout Mice, Circulation. 2003Sep. 9; 108(10):1232-7), inflammatory bowel disease (Crohn's disease),etc., as demonstrated in documented animal models and human clinicaltrials, as exemplified below.

In our adoptive cell transfer protocols, a mixed population of T cellsis initially extacted from a target donor. Depending on the application,the T cells may be extracted during a period of remission, or duringactive disease. Typically this is done by withdrawing whole blood andharvesting granulocytes by leukapheresis (leukopheresis). For example,large volume leukapherisis (LVL) has been shown to maximize bloodleukocyte yield. Harvests reach 20×10⁶ cells/L using a continuous flowapheresis device (Spectra, COBE BCT). Symptoms of hypocalcemia areavoided by a continuous infusion of calcium administrated throughoutleukapheresis. Typically 15-45 liters of fluid corresponding to about 4total blood volumes are harvested during a period of time ranging fromabout 100 to 300 minutes.

The harvested lymphocytes may be separated by flow cytometry or othercell separation techniques based on Treg-specific cell markers such asCD4, CD25 and CD62, expanded as described herein, and then transfused toa patient, typically the cell donor (except in GVHD where the donor andrecipient are different), for adoptive immune suppression.Alternatively, the cells may be frozen for storage and/or transportprior to and/or subsequent to expansion. For antigen non-specificexpansions, approximately 10⁹ to 10¹¹ Tregs are transfused; forantigen-specific expansions, therapeutically effective transfusionstypically require about 10⁷ to 10⁹ Treg cells.

Graft Versus Host Disease (GVHD). Ex vivo expanded CD4⁺CD25+cellsinhibit GVHD generation in our experimental protocol adapted fromTaylor, et al. Blood 99, 3493-9 (2002). 2×10⁶ freshly purified B6 CD4⁺ Tcells plus 5×10⁶ bone marrow cells are infused into irradiated,BALB/c×B6(F1) recipients. Cohorts of mice receive a separate injectionof 2×10⁶ activated CD4⁺CD25+cells or CD4⁺CD25⁻ cells, and survival andweights are monitored. The infusion of ex vivo-expanded CD4⁺CD25+cellssignificantly increases the median survival time from 10 days to greaterthan 100 days in 80% of mice. Survival in mice receiving supplementalexpanded CD4⁺CD25⁻ cells is not significantly different from controlmice receiving only fresh CD4⁺ T cells, indicating that the protectiveeffect is specific to the expanded CD4⁺CD25+population. Similar resultsare obtained using fresh (nonexpanded) donor-derived CD4⁺CD25+Treg cellsto prevent GVHD lethality in an experimental protocol adapted fromEdinger, et al. Nat Med 9, 1144-50 (2003). Animals that receive ex vivoexpanded Treg cells at a 1:1 ratio with conventional cells are protectedfrom acute lethal GVHD, and >80% survive more than 100 days.

Graft-versus-tumor (GVT) activity of Tconv cells is also maintainedafter cotransplantation of expanded Treg cells. GVHD is evaluated byclinical features and survival, tumor growth and rejection. A20-luc/yfpcells injected at the time of BMT migrate to the bone marrow, resultingin leukemia with secondary infiltration of liver and lymphoid organs(Edinger, et al. Blood 101, 640-648 (2003). BALB/c mice transplantedwith TCD BM from C57BL/6 animals and coinjected with 1×10⁴ A20-luc/yfpleukemia cells die before day 36 from leukemia, as demonstrated by anincrease in bioluminescence imaging (BLI) signal intensity over time.BLI images show that tumor cells infiltrate the bone marrow of humerus,femur and sternum 5 d after transplantation and additional organs,including the spleen, before day 15. Animals that receive TCD BM andTconv cells die even earlier from GVHD, but show initial engraftment ofthe A20-luc/yfp leukemia with a tumor cell distribution similar to thatof the bone marrow only control group. In contrast, the majority ofanimals receiving Tconv cells with CD4⁺CD25⁺ Treg cells survive theobservation period of 60 d. None of the animals show growth of leukemia,although all showed an initial tumor signal from the bone marrow at day5, demonstrating that T-cell transplantation does not interfere with theengraftment of A20-luc/yfp cells, but that an active eradication ofleukemia cells is achieved when animals are protected from lethal GVHDby donor CD4⁺CD25⁺ Treg cells. These data demonstrate that GVHDsuppression by our expanded CD4⁺CD25⁺ Treg cells does not abrogate GVTactivity of adoptively transferred donor Tconv cells.

Multiple Sclerosis (MS). Numerous studies have suggested that loss ofTreg cell is responsible for the lack of immunoregulation observed inpatients with MS (e.g. Putheti et al., Eur J Neurol. 2003 September;10(5):529-35; Baecher-Allan et al., J Immunol, 167:1245-53. 2001;Baecher-Allan et al, J. Immunol. 2002. 169(11):6210-7; Schmied, et al.,Clin Immunol. 2003 March; 106(3):163-74), and that adoptive cell therapymay ameliorate disease (e.g. Muraro et al. Immunological questions onhematopoietic stem cell transplantation for multiple sclerosis, BoneMarrow Transplant. 2003 August; 32 Suppl 1:S41-4; Blevins et al. Futureimmunotherapies in multiple sclerosis, Semin Neurol. 2003 June; 23(2):147-58; Kohm, et al., Cutting Edge: CD4⁺CD25⁺ Regulatory T CellsSuppress Antigen-Specific Autoreactive Immune Responses and CentralNervous System Inflammation During Active Experimental AutoimmuneEncephalomyelitis, J. Immunol., Nov. 1, 2002; 169(9): 4712-4716. Eur JNeurol. 2003 September; 10(5):529-35).

In our initial studies of adoptive immunosuppression therapy with MSpatients, T cells are harvested at time of remission, and Tregs areexpanded with anti-CD3 and anti-CD28 antibodies (supra), andfreeze-stored. The expanded Tregs are infused by injection at time ofrelapse, and disease progression is monitored using gadolinium-enhancedlesions (e.g. Horsfield et al., Guidelines for using quantitativemagnetization transfer magnetic resonance imaging for monitoringtreatment of multiple sclerosis, J Magn Reson Imaging. 2003 April;17(4):389-97). In subsequent studies, antigen-specific expansion iseffected using DR2 MHC coupled with immunogenic peptides of myelin basicprotein (MBP), myelin oligodendroctye glycoprotein (MOG), proteolipidprotein (PLP). Separation, expansion and freeze storage is carried outas above, except that expansion is effected with MHC peptide multimers(supra) plus anti-CD28 antibody and IL-2.

Rheumatoid Arthritis (RA). Prior studies have suggested that loss ofTreg cell is responsible for the lack of immunoregulation observed inpatients with RA, and animal models for therapeutic intervention havebeen validated. For example, a particular animal model of chronicinflammatory arthritis, ovalbumin-induced arthritis (OIA) has been usedto specifically analyze the role of defined populations ofantigen-specific T cells; see, Hardung, Regulatory function ofantigen-specific T helper cell subsets in a murine arthritis model, Proc34th Ann Meet German Soc Immunol, Berlin, Sep. 24-27, 2003. In thissystem, transfer of activated antigen-specific T helper cells(Ova-TCR^(tg/tg)) into naive, congenic recipients was sufficient toinduce joint inflammation after intraarticular injection of the antigen.Transfer of Th1 cells polarized in vitro resulted in an acute andchronic joint inflammation. Furthermore co-transfer of Ova-TCR^(tg/tg)CD4⁺CD25⁺ regulatory T cells prevented the induction of the disease.

In our initial studies of adoptive immunosuppression therapy with RApatients, T cells are harvested at time of remission from PBMC or fromjoint synovial fluid (Cao et al. Isolation and functionalcharacterization of regulatory CD25^(bright)CD4+ T cells from peripheralblood or the target organ of patients with rheumatoid arthritis. Eur J.Immunol. 2003 January; 33(1):215-23) and Tregs are expanded withanti-CD3 and anti-CD28 antibodies (supra) and IL-2, and freeze-stored.The expanded Tregs are infused by injection at time of relapse, anddisease progression is monitored using established clinical criteria(Felson et al, The American College of Rheumatology preliminary core setof disease activity measures for rheumatoid arthritis clinical trials.The Committee on Outcome Measures in Rheumatoid Arthritis ClinicalTrials. Arthritis Rheum 1993 June; 36(6):729-40; Felson et al., AmericanCollege of Rheumatology. Preliminary definition of improvement inrheumatoid arthritis, Arthritis Rheum 1995 June; 38(6):727-35).

In subsequent studies, antigen-specific expansion is effected using DR4MHC coupled with peptides for RA-associated autoantigens such asheat-shock proteins (HSPs), MHC-derived peptides, and joint-specificantigens such as type II collagen (e.g. Kotzin, Use of solublepeptide-DR4 tetramers to detect synovial T cells specific for cartilageantigens in patients with rheumatoid arthritis, Proc Natl Acad Sci USA2000 Jan. 4; 97(1):291-6). Separation, expansion and freeze storage iscarried out as above, except that expansion is effected with MHC peptidemultimers (supra) plus anti-CD28 antibody and IL-2.

Psoriasis. Prior studies have suggested that loss of Treg cell isresponsible for the lack of immunoregulation observed in patients withpsoriasis, and animal models for therapeutic intervention has beenvalidated (see, e.g. Elder et al., Of genes and antigens: theinheritance of psoriasis. J Invest Dermatol. 1994 November; 103, 5Suppl, 150S-153S).

In our initial studies of adoptive immunosuppression therapy withpsoriasis patients, T cells are harvested at time of remission fromPBMC, or during active disease from skin and Tregs are expanded withanti-CD3 and anti-CD28 (supra) antibodies and IL-2, and freeze-stored.The expanded Tregs are infused by injection at time of relapse, anddisease progression is monitored using established clinical criteriawherein the primary clinical end point is the mean percentage change inthe PASI score comparing baseline and week 12 scores (see, e.g. Ashcroftet al., Clinical measures of disease severity and outcome in psoriasis:a critical appraisal of their quality. Br J. Dermatol. 1999 August;141(2):185-91).

In subsequent studies, antigen-specific expansion is effected using DR6MHC coupled with peptides of psoriasis-associated skin autoantigens, andin the case of psoriatic arthritis, joint autoantigens. Separation,expansion and freeze storage is carried out as above, except thatexpansion is effected with MHC peptide multimers (supra) plus anti-CD28antibody and IL-2.

Inflammatory Bowel Disease (IBD), Crohn's Disease, colitis. Priorstudies have suggested that loss of Treg cell is responsible for thelack of immunoregulation observed in patients with IBD, and animalmodels for therapeutic intervention has been validated (see, e.g.Assessman et al., Colitogenic Th1 cells are present in theantigen-experienced T cell pool in normal mice: control by CD4+regulatory T cells and IL-10. J. Immunol. 2003 Jul. 15; 171(2):971-8;Read, et al., 1998. CD38⁺CD45RB^(nlow)CD4⁺ T cells: A T cell populationwith immune regulatory activities in vitro. Eur. J. Immunol. 28:3435;Mason et al, 1998. Control of Immune Pathology by regulatory T cells.Curr. Opin. in Immunol. 10:649; Asseman, et al., 1999. IL-10 is requiredfor the generation of a population of T cells, which regulateinflammatory responses in the intestine. J. Exp. Med. 190:995).

In our initial studies of adoptive immunosuppression therapy with IBDpatients, T cells are harvested at time of remission from PBMC, orduring active disease from affected intestinal tissue, and Tregs areexpanded with anti-CD3 and anti-CD28 antibodies (supra) and IL-2, andfreeze-stored. The expanded Tregs are infused by injection at time ofrelapse, and disease progression is monitored using established clinicalcriteria correlated with histologic evidence of gastroenteritisincluding moderate to severe infiltrates of inflammatory cells, chronicintermittent, long duration diarrhea, weight loss, vomiting, etc.,wherein alternative sources of intestinal inflammation have beenclinically excluded.

In subsequent studies, antigen-specific expansion is effected usingHLA-CW6 coupled with peptides of dietary and/or bacterial antigensassociated with IBD hypersensitivity. Separation, expansion and freezestorage is carried out as above, except that expansion is effected withMHC peptide multimers (supra) plus anti-CD28 antibody and IL-2.

The invention provides Treg cell compositions made by the subjectmethods, particularly compositions adapted for transfusion into patientsin need of autoimmune suppression, as described herein. For example,such compositions include effective transfusable unit dosages ofexpanded Treg cells as described herein, wherein such dosages may beprepackaged with in kits as described below.

The invention provides kits comprising reagent(s) and/or material(s) foruse in a subject method, and optionally, an instructional mediumdescribing a subject method. The invention also provides businessmethods specifically adapted to, and/or incorporating a description of,or reference to a subject method or kit.

ADDITIONAL EXAMPLES Example 1 In Vitro Expanded Antigen-Specific TregCells Suppress Autoimmune Diabetes

Here we describe a robust method to expand antigen-specific Tregs fromautoimmune-prone non-obese diabetic (NOD) mice. The Tregs, expanded upto 200-fold in less than 2 weeks in vitro, express a classical Tregphenotype, retaining all the quintessential characteristics of thissubset including expression of CD25, CD62L, FoxP3, and GITR, andfunction both in vitro and in vivo to suppress effector T cellfunctions. The ability of expanded NOD Tregs to suppress diabetes inprediabetic and diabetic mice in vivo was significantly enhanced usingthe autoantigen-specific T cells when compared to polyclonal Tregs.Antigen-specific Tregs effectively suppressed the development ofdiabetes in Treg-deficient CD28^(−/−) mice, blocked syngeneic isletgraft rejection in chronically diabetic animals and, in contrast toprevious reports, Tregs are shown, for the first time, to reversediabetes in mice with new onset disease. Hence, this is the firstdemonstration that small numbers of antigen-specific Tregs can reversediabetes following disease onset, providing a novel approach to cellularimmunotherapy for autoimmunity.

Expansion of Regulatory T cells from autoantigen-specific TCR transgenicNOD mice. Previous studies have shown that Tregs decrease in number andfunction in NOD mice over time correlating with clinical disease by16-24 weeks of age. However, the ability to use these cellstherapeutically is contraindicated by the small numbers of cellsresident in the circulation or lymphoid organs (<5% of CD4⁺ T cells inNOD mice and <2% of CD4⁺ T cells in humans with T1D). Moreover, a largenumber of cells are required due to difficulty selecting the cells basedon antigen specificity. Therefore, we developed a technique for rapidand efficient expansion of autoantigen-specific Tregs based onobservations that these cells, present in TCR transgenic (Tg) mice, canbe driven into cell cycle with co-immobilized anti-CD3 and anti-CD28antibodies plus exogenous IL-2. FACS-purified NOD Tregs cultured withanti-CD3/anti-CD28-coated beads in the presence of IL-2 expanded 150-225fold in 11 days. In general, the CD4⁺CD25⁻ T cells expanded morevigorously (ranging from 300-800-fold in multiple experiments). Thus, apurity of >98% CD4⁺CD25⁺CD62⁺ T cells was preferred for successful Tregexpansion as a small contamination of either CD25⁻CD4⁺ or CD8⁺ T cellsimpacted the ability to expand the Tregs.

Previous studies have reported that CD4⁺CD25⁺ Tregs, isolated from youngNOD mice suppressed the ability of Teff cells from diabetic NOD mice totransfer disease in immunodeficient NOD mice. However, the process wasinefficient and the suppressive effects of Tregs in this settingrequired a 0.5:1 or 1:1 ratio of Treg:Teff, likely due to the lowprecursor frequency of antigen-specific Tregs. Thus, we examined whetherTregs from two different antigen-specific TCR Tg mice could be expandedin vitro using the same methodology as with the polyclonal NOD Tregs.BDC2.5 TCR Tg mice express a TCR specific for an islet antigen expressedin the granules of β cells while the GAD286 TCR Tg recognizes a peptidederived from the islet antigen, glutamic acid decarboxylase (GAD). Tregswere purified from BDC2.5 and GAD286 mice and expanded using theanti-CD3/anti-CD28 plus IL-2 cocktail. The BDC2.5 cells expressed thetransgenic TCRαβ based on efficient staining with a MHC-peptide tetramerpreviously shown to react with this TCR and the expanded GAD286 Tregsexpressed the Tg TCRβ chain. The CD4⁺CD62L+CD25⁻ and Tregs from BDC2.5TCR Tg mice can be expanded at similar efficiency using immobilizedMHC-peptide dimers. These results indicate that a population ofCD4⁺CD25⁺CD62L+exist in both wild type and TCR Tg mice that can beexpanded using this protocol.

We next examined the phenotype of the expanded Tregs by flow cytometry,western blot and real time PCR. The expanded Tregs maintained highlevels of expression of CD25 as compared to expanded CD25-T cells,whereas the expression of CD62L remained high in both cell types. Inaddition, quantitative PCR showed that all the Tregs expressed highlevels of SOCS2, PD-1, and CTLA-4 as compared to similarly expandedCD25⁻ T cells. Moreover, the recently identified markers neuropilin andTRAIL were also highly expressed on the expanded Tregs. A high level ofcell surface GITR expression was observed on the expanded Tregs,however, this previously identified Treg marker was also induced on theexpanded CD25⁻ T cells. It should be noted that the quantitative PCRstudies were performed on five separate expanded Treg populations(including both polyclonal and BDC2.5 TCR Tg Tregs) and the relativeexpression was highly reproducible. Finally, we examined the recentlyidentified lineage marker for Tregs, FoxP3. As noted by both RT-PCR andwestern blot analyses, the expanded Tregs expressed levels of FoxP3similar to that observed in fresh Tregs and significantly higher thanfresh or expanded CD25⁻T cells. The RNA expression (10-fold) and proteinamounts (20-fold) were consistent with previous studies of fresh Tregsalthough there was clearly some increase in FoxP3 in CD25⁻Teff cellsindicating that the culture conditions may induce some regulatory Tcells within the CD25⁻ subset.

We also examined the ability of the expanded Tregs to secrete cytokines.Unlike activated CD25⁻T cells, the Tregs did not produce IL-2 or IFNγbut rather expressed the immunosuppressive cytokines IL-10 and TGFβ.Thus, the extensive activation and proliferation of the Tregs does notalter the phenotype of the Tregs which remained distinct from the CD25⁻T cell subset.

Functional activity of in vitro expanded Tregs. Previous studies haveshown that Tregs can effectively suppress proliferative responses ofCD25⁻T cells stimulated with anti-CD3 and splenic APC. The expanded NODTregs efficiently suppressed proliferative responses and cytokineproduction including IL-2 and IFNγ. In fact, in multiple experiments,the expanded Tregs suppressed significantly better than fresh NOD Tregs.The suppression was routinely observed at Treg:Teff ratios of <1:10.Similar results were observed using the expanded Tregs from the TCR Tgmice as the expanded BDC2.5 Tregs were effective in suppressing theproliferative response of BDC2.5 as well as polyclonal NOD T cells.Examination of the mechanism of Treg suppression confirmed other studiesdemonstrating a requirement for cell-cell contact. Although the expandedTregs expressed significant levels of IL-10 and TGFβ, suppressoractivity was unaffected by the addition of anti-IL-10, anti-TGFβ or acombination of both antibodies to the in vitro cultures. These resultsare consistent with numerous models of Treg suppression where cell-cellcontact is the primary means of immunosuppression in the in vitrosetting.

To further assess the antigen-specificity of the expanded Tregs anddetermine whether the expanded Tregs were constitutively suppressive,expanded Tregs from normal BALB/c mice were examined for their abilityto suppress T cells from the OVA-specific DO11.10 TCR Tg mouse. Tregsand DO11.10 Tg Teff cells were co-cultured in the presence of OVAantigen (to activate only the Teff cells) or anti-CD3 (to activate boththe Teff and Tregs). Expanded BALB/c Tregs did not inhibit theproliferative response of the DO11.10 T cells stimulated by the OVApeptide. However, the anti-CD3 response was fully inhibited at lowTreg:Teff ratios supporting the lack of constitutive suppressiveactivity of the expanded Tregs and the requirement for antigen-specificactivation of Tregs for effective immune suppression. This result alsoruled out the trivial possibility that the cells were inhibiting thecultures by consuming available IL-2 through the high level of CD25expression.

In vivo survival and activation of expanded Tregs. Effective suppressionof immune responses in vivo by Tregs requires that the cells migrate toappropriate sites, respond to antigen and survive long term. We haveobserved recently that blockade of the CD28/B7 pathway resulted in rapidloss of Tregs in vivo and subsequent loss of critical immune regulation.Thus, we examined the ability of expanded Tregs to survive andproliferate in vivo. Expanded Tregs from NOD, BDC2.5 and GAD286 micewere labeled with CSFE and transferred into normal non-lymphopenic NODmice. At 7 days post transfer, the mice were sacrificed and examined forthe number of CSFE⁺ cells as a read out of survival and proliferation. Asignificant number of CSFE⁺ cells were recovered from mice transferredwith expanded Tregs from the different mouse strains. In fact, CFSE⁺Tregs as well as Thyl 0.1-marked Tregs were observed at least 50 dayspost transfer, and the numbers were equal to those observed with freshTregs transferred in the same manner.

Next, we analyzed the ability of the adoptively transferred Tregs torespond to antigen and to proliferate in vivo. To obviate the potentialfor lymphopenia-driven proliferation, the Tregs were transferred intonormal mice. A small, but significant number of Tregs, proliferatedbased on CSFE dilution. However, there was no selective proliferation ofthe NOD Tregs in the pancreatic lymph nodes (pancLN) indicating thatthere was not a significant number of islet autoantigen-specific cellswithin the NOD Treg repertoire. In fact, there were fewer expanded Tregsin the NOD pancLN cells than observed in other LN cells, indicating thepossibility that islet-specific Tregs were deleted in the NOD. Incontrast to the NOD Tregs, Tregs from BDC2.5 Tg mice proliferated andexpanded extensively and selectively in the pancLN dividing at least 3-4times during the 7 day period. Interestingly, the proliferating Tregsdown-regulated CD62L expression. This is surprising since the cells hadundergone multiple proliferative cycles in vitro prior to transfer andhad maintained high levels of CD62L expression. In contrast to theBDC2.5 Tregs, the GAD286 Tregs did not proliferate in vivo. Previousstudies suggest that these two TCR Tg mice differ significantly in theirthymic development. The BDC2.5 Tg mice do not negatively select theislet specificity in the thymus but rather develop a small butreproducible number of Tregs that have been shown to block disease bypotential effector cells resident in these animals. By comparison,GAD286 TCR Tg T cells are negatively selected in the thymus such thatthe cells that escape utilize alternative TCRα chains. Although theperipheral GAD286 TCR Tg cells respond to GAD peptide in vitro, thereactivity is weak and, in contrast to the BDC2.5, are unable to inducediabetes upon adoptive transfer indicating the “absence” of anautoreactive repertoire. Thus, these results using the expanded Tregsindicate that the BCD2.5 TCR Tg, but not GAD286 TCR Tg mice havecirculating autoreactive Tregs that home and survive in vivo and receiveadditional signals to further activate and expand the antigen-specificsubset.

In vitro expanded Tregs suppress adoptive transfer of diabetes in vivo.Next, we examined the ability of the expanded BDC2.5 Tregs to suppressdiabetes following in vivo co-transfer of activated BDC2.5 T cells intoNOD.RAG mice. The Tregs were effective in blocking the transfer ofdiabetes, functioning at as low as a 1:9 ratio of Treg:Teff, whereas theGAD286 Tregs did not protect even at Treg:Teff of 1:1. In fact, theexpanded BDC2.5 Tregs suppressed polyclonal T cell-mediated disease. Asfew as 2×10⁶ expanded BDC2.5 Tregs blocked the ability of 25×10⁶diabetogenic NOD spleen cells to transfer disease. The expandedantigen-specific Tregs from the BDC2.5 mice were far more efficient thanexpanded polyclonal NOD Tregs in preventing the onset of diabetes,consistent with the distinct proliferative differences described above.As many as 8×10⁶ expanded NOD Tregs prevented diabetes in only 25% ofdiabetogenic cell transferred RAG recipients as compared to totalblockade of disease transfer using one quarter the number of theantigen-specific BDC2.5 Tregs. This result is consistent with previousfindings suggesting that a high ratio of polyclonal Treg to Teff cellsare necessary to efficiently suppress disease transfer in this setting.Importantly, these data indicate that in vitro reactivity of the Tregsdoes not predict in vivo function in this disease.

Expanded Tregs prevent diabetes in vivo in a non-lymphopenic setting.Although there are multiple models demonstrating the immunoregulatoryactivity of Tregs, many of the systems are based on adoptive transfermodels that take advantage of lymphopenic mice to enhance Tregproliferation.^(8,21-24) Therefore, we examined the ability of theexpanded Tregs to prevent diabetes in a non-lymphopenic animal model.Previous studies have shown that CD28^(−/−) NOD mice have normal numbersof T cells and Th1 responses. In fact, these mice develop exacerbatedautoimmunity due to a deficiency in Th2 and Tregs which were shown to beexquisitely CD28-dependent. Thus, we examined whether wild type expandedBDC2.5 Tregs could be transferred into CD28^(−/−) NOD mice and delay orprevent onset of disease. Five×10⁵ Tregs were transferred into 5 weekold CD28^(−/−) NOD mice and monitored for diabetes. The transfer ofexpanded BDC2.5 Tregs prevented the development of diabetes in 100% ofmice examined as long as 20 weeks after transfer. In contrast, thetransfer of expanded NOD Tregs had no effect on disease incidence. Theseresults indicate that the antigen-specific expanded Tregs functioned invivo in the face of a fully functional pathogenic T cell response.

Expanded Tregs reverse diabetes in vivo. The ultimate utility of Tregtherapy depends on being able to treat individuals with ongoing disease.Thus, we extended an examination of the regulatory effects of theseexpanded Tregs in frank models of diabetes. First, we examined theability of expanded BDC2.5 Tregs to block rejection of a syngeneic NODislet transplant. Normoglycemia was maintained in diabetic NOD miceusing insulin pellets for at least two weeks. At that time, the micewere transplanted with 500 syngeneic islet cells alone or in conjunctionwith expanded Tregs. The co-transfer of 2×10⁶ BDC2.5, but not 5×10⁶ NOD,expanded Tregs blocked rejection of the syngeneic islets consistent withan ability of the suppressor cells to block ongoing autoimmunity in thissetting. More significantly, the adoptive transfer of expanded BDC2.5Tregs reversed diabetes in overtly diabetic NOD mice. In this setting,1×10⁷ Tregs were transferred into NOD mice diagnosed with recent diseaseonset based on elevated blood glucose levels (>300 mg/dL). Thetransferred Tregs reversed diabetes in 60% of the mice. Thus, theexpanded Tregs were extremely effective in blocking and reversingdiabetes in an ongoing autoimmune setting.

Our observation that the Tregs are able to reverse diabetes demonstratesthe applicability of our methods for clinical autoimmune therapy, whereTregs are isolated from patients either during remission (e.g for SLE orMS) or soon after disease onset (e.g. for T1D). The cells are thenexpanded and reintroduced at the time of maximal disease activity tomoderate the inflammatory response. We have found that this therapy canbe combined with Rapamycin, Anti-CD3 or other drugs that cause deletionof the pathogenic cells without affecting the Tregs. Together thesetherapies both reduce the short term pathogenic responses whilereinstating a homeostatic balance for long-term tolerance induction.

Example 2 Expansion of Functional Endogenous Antigen-Specific CD4⁺CD25⁺Regulatory T Cells from NOD Mice: Antigen-Specific CD4⁺CD25⁺ RegulatoryT Cells Control Autoimmune Diabetes

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) are critical for controllingautoimmunity. Evidence suggests Treg development and function aredependent on antigen specificity. Despite this, little is known aboutantigen-specific Tregs arising in natural settings. In this example weidentify and characterize Tregs that recognize an islet peptide-mimicand arise naturally in nonobese diabetic mice. Antigen-specific Tregsexpress prototypic surface markers and cytokines. Although activated inan antigen-specific fashion, the expanded Tregs were capable ofbystander suppression both in vitro and in vivo. Importantly, the isletpeptide mimic-specific Tregs were more efficient than polyclonal Tregsin suppressing autoimmune diabetes. Our disclosure demonstrates theutility of Tregs as therapeutics for organ-specific autoimmunity.

Autoimmune type 1 diabetes (T1D) develops due to a breakdown in themechanisms responsible for maintaining tolerance to self-antigens,resulting in T cell-mediated destruction of the insulin-producing isletcells of the pancreas. Potentially pathogenic self-reactive T cells arepresent in the normal peripheral T cell repertoire but in healthyindividuals are controlled in part by suppressor or regulatory T cells(Tregs) (1, 2). Among the classes of Tregs, CD4⁺CD25⁺ Tregs are a uniquecell subset important for controlling autoimmunity (3, 4). Mice andhumans deficient in CD4⁺CD25⁺ Tregs or Foxp3, the transcription factorthat controls CD4⁺CD25⁺ Treg development and suppressor function, sufferfrom multiorgan autoimmune disease (5-11). A decreased or impairedsuppressor function of CD4⁺CD25⁺ Tregs has been associated with TID,multiple sclerosis, rheumatoid arthritis, and other autoimmune diseases(12-16). By comparison, the transfer of polyclonal CD4⁺CD25⁺ regulatoryT cells prevented autoimmunity in a number of systems includingautoimmune diabetes in nonobese diabetic (NOD) mice, a mouse model ofTID (17, 18). However, the process was inefficient and required thetransfer of high numbers of Tregs. We recently described a method forthe in vitro expansion of islet antigen-specific CD4⁺CD25⁺ BDC2.5 T cellreceptor transgenic (TCR Tg⁺) Tregs using a combination of IL-2 andbeads coated with anti-CD3 and anti-CD28 mAb (19). The expandedislet-specific Tregs were effective in blocking and reversing diabetesin NOD mice using significantly reduced numbers of Tregs as compared topolyclonal NOD Tregs; indicating that antigen-specificity of the Tregsis important for therapeutic efficacy. Therefore, effective clinicaltherapy depends on the ability to identify and expand relevantantigen-specific Tregs from polyclonal populations (20).

Relatively little is known about the antigen-specificity of CD4⁺CD25⁺Tregs arising under natural conditions. In the present study, wehypothesized that since BDC2.5 TCR Tg⁺ mice have a significantpercentage of islet antigen-specific Treg this specificity might bepresent in conventional NOD mice as well (19, 21, 22). Thus, we adaptedthe expansion protocol used in the BDC2.5 Treg studies by substitutingthe anti-CD3 mAb with a recombinant MHC class I11-A^(g7) presenting theBDC2.5 TCR mimotope peptide 1040-31 (p31) (23). The mimotope peptide wasused because the endogenous BDC2.5 antigen is not yet identified (24).To determine if p31-I-A^(g7) beads could expand low frequencyantigen-specific cells from a polyclonal population BDC2.5 TCR Tg⁺ Tregswere seeded into polyclonal CD4⁺CD25⁺ Treg cells from NOD mice. Thep31-I-A^(g7) and anti-CD28 coated beads were extremely efficient inexpanding CD4⁺CD25⁺ BDC2.5 TCR Tg⁺ Tregs in the presence of exogenousIL-2. Cultures initially seeded at 0.1% BDC2.5 TCR Tg⁺ Tregs expandedapproximately 4-fold, whereas cultures seeded at 0.01% and 0.001% BDC2.5TCR Tg⁺ Tregs did not expand appreciably. However, flow cytometryanalysis using p31-I-A^(g7) multimers to detect antigen-specific cellsrevealed that BDC2.5 TCR Tg⁺ Tregs had expanded in all cultures. At thelowest seeding, BDC2.5 TCR Tg⁺ Tregs grew from 0.001% to 34.3% of thepopulation. This reflected greater than 12 cell divisions during theculture period resulting in nearly a 5000 fold expansion of theantigen-specific cells during the 10 day culture. To ensure thatCD4⁺CD25⁺ Tregs retained regulatory activity after expansion withpeptide-I-A^(g7) coated beads, suppression assays were performed usingfreshly isolated CD4⁺CD25⁻ BDC2.5 Tg⁺ responder cells in combinationwith a titration of expanded CD4⁺CD25+BDC2.5 Tg⁺ Tregs. ExpandedCD4⁺CD25⁺ BDC2.5 Tregs efficiently suppressed the CD4⁺CD25⁻ T cellresponse in a dose dependent manner in cultures stimulated with theBDC2.5 mimotope peptide 1040-31. Furthermore, suppressive activity wasnot lost after multiple rounds of in vitro stimulation. CD4⁺CD25⁺ BDC2.5Tg⁺ cells, initially seeded at 0.001% and expanded to 50% after tworounds of stimulation with peptide-I-A^(g7) beads, suppressed CD4⁺CD25⁻BDC2.5 Tg⁺ cells stimulated with the p31 peptide. Thus, even when theantigen-specific BDC2.5 TCR Tg+ Treg cells represented an extremelysmall percentage of the total polyclonal Treg population, the procedureresulted in a large expansion of antigen-specifc Tregs that retainedsuppressive function.

We next applied this approach to the expansion of antigen-specificCD4⁺CD25⁺ Tregs from conventional NOD mice. CD4⁺CD25⁺CD62L⁺ cells fromNOD mice were cultured with p31-I-A^(g7) beads as described in Materialand Methods (25). Over a 7 to 14 day period the total populationtypically expanded 1 to 10 fold compared to the initial cell input. Flowcytometry analysis demonstrated that after expansion with thep31-I-A^(g7) beads up to 10% of CD4⁺CD25⁺ cells stained positive for thep31-I-A^(g7) multimer while CD4⁺CD25⁺ cells expanded withanti-CD3-coated beads did not stain positive for p31-I-A^(g7) abovebackground levels. Under the same culture conditions CD4⁺CD25⁻CD62L⁺ Teffectors (Teff) typically expanded 10 fold with 40 to 50% stainingpositive for the p31-I-A^(g7) multimer. However, the multimer stainingwas clearly an underestimate of the p31-I-A^(g7)-reactive Treg cellsbased on in vitro proliferation assays. Expanded Treg cells were labeledwith carboxyfluorescein succinimidyl ester (CFSE) and cultured with thep31 peptide or control ovalbumin (OVA) peptide in the presence ofantigen presenting cells (APC) and anti-CD28. The results of CFSEdilution assays showed that over 50% of p31-I-A^(g7) cultured Tregsentered into cell cycle compared to the background proliferation of 14%in ovalbumin-stimulated cultures. The high degree of backgroundproliferation seen with the OVA peptide may reflect that the cultureswere not 100% resting due to the continual presence of beads in theculture. Prior to stimulation, this same cell population had only 6.4%p31-reactive cells when analyzed by flow cytometry for p31-I-A^(g7)multimer binding. These results indicate that T cells with low avidityare poorly detected by multimer staining and may reflect the fact thatthe TCR are specific for endogenous antigens that are not precisely thesame as the p31 mimotope of the BDC2.5 specificity. To explore thevailidity of this interpretation we examined the VP repertoire of thep31-I-A^(g7)-expanded T cells. The BDC2.5 T cell receptor expresses aTCRP derived from the Vβ4 family (26). However, when p31-I-A^(g7)expanded Treg and Teff cells were co-stained with p31-I-A^(g7) multimersand different TCR VP reagents neither population was monoclonal.Instead, both populations showed a broad repertoire with several VPpopulations represented. Interestingly, although there were asignificant number of Vβ4⁺ p31-I-A^(g7) multimer⁺ T cells in the Tregculture, other TCR VP were also present in significant numbers, forinstance, Vβ2 and Vβ12 which accounted for 10.2 and 13.7% of thep31-I-A^(g7) multimer+Tregs, respectively, in this representativeculture. TCR Vβ4⁺ T cells were generally present in the p31-I-A^(g7)multimer⁺ T effector population but at a lower percentage. Togetherthese results indicate that a broad repertoire of Treg and Teff cellsreactive against the islet peptide-mimic are resident in conventionalNOD mice. The results also indicate that the isletpeptide-mimic-reactive Treg repertoire is not identical to the isletpeptide-mimic-reactive Teff repertoire.

As observed previously for anti-CD3-expanded cultures,peptide-I-A^(g7)-expanded Tregs retained the CD4⁺CD25⁺CD62L⁺ phenotypethroughout the culture period in contrast to CD4⁺CD25-CD62L⁺ cells thatwere cultured in a similar manner. CD4⁺CD25⁻CD62L⁺ cells became CD25highupon activation but after the initial activation slightly down regulatedCD25 compared to p31-I-A^(g7) reactive Treg cells. The majority ofp31-I-A^(g7)-reactive T effector cells down regulated CD62L during theculture period. We also examined the expanded Treg and Teff cells forthe expression of the Treg lineage marker Foxp3 using quantitative realtime PCR. To ensure that p31-I-A^(g7) reactive cells were analyzed,p31-I-A^(g7)-expanded Tregs were sorted into p31-I-A^(g7)-multimerpositive and negative populations by fluorescent activated cell sorting(FACS) prior to analysis. In a representative experiment expandedp31-I-A^(g7)-multimer⁺ Tregs expressed approximately 3000 fold moreFoxp3 relative to expanded p31-I-A^(g7)-multimer positive T effectors.Expanded p31-I-A^(g7) Tregs also expressed the quintessential Tregsurface markers CTLA-4, ICOS, and GITR when analyzed by flow cytometry.We next examined cytokine secretion by p31-I-A^(g7) expanded Tregs uponchallenge with antigen. Consistent with data reported previously forTregs, p31-I-A^(g7)-expanded Tregs expressed low levels of theproinflammatory cytokines IL-2. IL-4, and IFNγ and expressed high levelsof the anti-inflammatory cytokine IL-10.

Previous studies have shown that CD4⁺CD25⁺ Tregs can suppressproliferation of CD4⁺ effectors in vitro and that the suppressive effectis dependent on stimulation of CD4⁺CD25⁺ Tregs through their TCR.Therefore, expanded p31-I-A^(g7) Treg cells were examined forsuppressive activity and specificity in vitro. Expanded p31-I-A^(g7)effectively suppressed the proliferation of freshly isolated polyclonalCD4⁺ T cells and antigen-specific CD4⁺ BDC2.5 TCR Tg⁺ mice in adose-specific manner when cultures were stimulated with the polyclonalactivator anti-CD3. More importantly, p31-I-A^(g7) Tregs suppressed theproliferation of BDC2.5 TCR Tg⁺ CD4⁺ T cells when the cultures werestimulated with the 1040-31 peptide demonstrating specific suppressionby 1040-31 peptide reactive cells in the culture. In contrast, expandedCD4⁺CD25⁻p31-I-A^(g7) Teffs failed to suppress freshly isolated BDC2.5CD4⁺ T cells and resulted in augmentation of proliferation.Interestingly, the p31-I-A^(g7) expanded Treg but not Teff cells wereanergic to stimulation (both p31 peptide and anti-CD3) in the absence ofCD28 co-stimulation consistent with reports for freshly isolated Tregs.To further characterize the antigen specificity of the expanded Tregs,expanded polyclonal Tregs and p31-I-A^(g7) expanded Tregs were assessedfor the ability to suppress BDC2.5 TCR Tg⁺ or Glutamic AcidDecarboxylase peptide 286-specific (GAD286) TCR Tg⁺ CD4⁺ cells througheither polyclonal T cell activation via anti-CD3 or antigen-specific Tcell activation (27). Both polyclonal Tregs and p31-I-A^(g7) expandedTregs suppressed BDC2.5 TCR Tg⁺ responders when stimulated withanti-CD3, whereas, only the p31-I-A^(g7) expanded Tregs suppressedcultures stimulated with the BDC2.5 1040-31 peptide. Similarly, bothpolyclonal Tregs and p31-I-A^(g7)-expanded Tregs suppressed the responseof GAD286 TCR Tg⁺ CD4⁺ T cells when stimulated with anti-CD3. However,neither the polyclonal Treg population nor the p31-I-A^(g7)-expandedTreg population suppressed GAD286 TCR Tg⁺ responders when stimulatedwith the GAD(286-300) peptide. Most significantly, p31-I-A^(g7) expandedTregs were capable of suppressing GAD286 TCR Tg⁺ responders when theculture was stimulated with both the GAD(286-300) and the 1040-31peptide. Collectively these data demonstrate that the suppressiveactivity of the peptide-I-A^(g7)-expanded Tregs is dependent onantigen-specific stimulation through the TCR, although, once stimulatedwith cognate antigen, p31-I-A^(g7) expanded Tregs are capable ofexerting bystander suppression.

We then tested the ability of small numbers of p31-I-A^(g7)-expandedTregs to suppress polyclonal T cell-mediated diabetes in CD28^(−/−) NODmice. CD28^(−/−) NOD mice have normal numbers of effector T cells andTh1 responses and undergo an accelerated form of autoimmune diabetes dueto a deficiency in Tregs which are dependent on CD28 for homeostasis inthe periphery (17, 28). Previous studies have shown that transfer ofhigh numbers (8-20×10⁶) of polyclonal Tregs can delay or prevent theonset of diabetes (17). Thus, we examined whether p31-I-A^(g7)-expandedTregs transferred into CD28^(−/−) mice could prevent diabetes. Transferof as few as 1.8 to 2×10⁶ p31-I-A^(g7) expanded Tregs into 5 to 7-wk-oldmice prevented the development of diabetes in 55% of mice for as long as15 weeks of age. The transferred populations typically contained ˜10%p31-1-A^(g7) multimer⁺ cells based on flow cytometry, although theabsolute frequency was undoubtedly higher based on the CFSEproliferation assays. Therefore, we estimate that the transferredpopulations contained 10⁶ or less antigen-specific Treg cells which issubstantially less than similar studies performed with polyclonal NODTregs. Thus, the antigen-specific p31-I-A^(g7) expanded cells werehighly efficient in protecting the onset of diabetes induced by a fullyfunctional polyclonal T cell response. Moreover, suppression ofautoimmunity by the p31-I-A^(g7) expanded Tregs was organ-specific asanimals that had received p31-I-A^(g7) expanded Tregs and were protectedfrom diabetes at 15 and 16 weeks-of-age displayed a a higher degree oflymphocytic infiltration in the salivary and thyroid glands compared tonon-treated and polyclonal Treg treated mice that were diabetic andexamined at 8-10 weeks-of-age.

In this example, we demonstrate that antigen-specific CD4⁺CD25⁺ Foxp3⁺Tregs reactive to an islet-peptide mimic reside in the periphery ofdiabetes-susceptible NOD mice. Furthermore, we demonstrate thatantigen-specific cells can be selectively expanded in vitro from apolyclonal population and that these expanded Tregs retain phenotypicand functional characteristics of freshly isolated CD4⁺CD25⁺ Foxp3⁺Tregs. We show that in vivo, expanded antigen-specific Tregs are highlyefficient at controlling organ-specific autoimmunity. These resultssupport previous studies demonstrating that immune regulation byCD4⁺CD25⁺ Tregs is dependent on the antigen specificity of the Tregs andare not consistent with reports suggesting that Tregs function in anantigen non-specific fashion by competing for T cell niches (19, 22,29).

Our findings provide Treg-based approaches for clinical therapy, whichentail expansion of organ-specific Tregs from peripheral blood. Evenwhere small numbers of autoantigen-specific Treg with restrictedrepertoires are expanded, these cells can be clinically efficaciousbecause of the ability to suppress polyclonal T cell responses either bybystander cytokine production and/or recruitment of endogenousregulatory cells. Many organ-specific antigens have been identified thatcontribute to autoimmune diseases such as T1D and multiple sclerosis,and currently available human MHC multimer reagents can be employed toexpand human organ-specific Tregs for treatment of autoimmune diseases(2).

REFERENCES

-   1. S. Arif et al., J. Clin. Invest. 113, 451 (2004).-   2. N. A. Danke, et al. k, J. Immunol. 172, 5967 (2004).-   3. L. Chatenoud, B. Salomon, J. A. Bluestone, Immunol. Rev. 182, 149    (2001).-   4. S. Sakaguchi, Annu. Rev. Immunol. 22, 531 (2004).-   5. T. A. Chatila et al., J. Clin. Invest. 106, R75 (2000).-   6. C. L. Bennett et al., Nature Genet. 27, 20 (2001).-   7. M. E. Brunkow et al., Nature Genet. 27, 68 (2001).-   8. R. S. Wildin et al., Nature Genet. 27, 18 (2001).-   9. J. D. Fontenot, M. A. Gavin, A. Y. Rudensky, Nature Immunol. 4,    330 (2003).-   10. R. Khattri, T. Cox, S. A. Yasayko, F. Ramsdell, Nature Immunol.    4, 337 (2003).-   11. S. Hori, T. Nomura, S. Sakaguchi, Science 299, 1057 (2003).-   12. A. Kukreja et al., J. Clin. Invest. 109, 131 (2002).-   13. M. R. Ehrenstein et al., J. Exp. Med. 200, 277 (2004).-   14. M. A. Kriegel et al., J. Exp. Med. 199, 1285 (2004).-   15. V. Viglietta, et al. J. Exp. Med. 199, 971 (2004).-   16. C. Baecher-Allan, D. A. Hafler, J. Exp. Med. 200, 273 (2004).-   17. B. Salomon et al., Immunity 12, 431 (2000).-   18. S. Gregori, N. Giarratana, S. Smiroldo, L. Adorini, J. Immunol.    171, 4040 (2003).-   19. Q. Tang et al., J. Exp. Med. 199, 1455 (2004).-   20. J. A. Bluestone, Q. Tang, Proc. Natl. Acad. Sci. U.S.A. 101,    14622 (2004).-   21. A. E. Herman, G. J. Freeman, D. Mathis, C. Benoist, J. Exp. Med.    199, 1479 (2004).-   22. K. V. Tarbell, et al., J. Exp. Med. 199, 1467 (2004).-   23. E. L. Masteller et al., J. Immunol. 171, 5587 (2003).-   24. V. Judkowski et al., J. Immunol. 166, 908 (2001).-   25. Information on materials and method is available on Science    Online.-   26. J. D. Katz, B. Wang, K. Haskins, C. Benoist, D. Mathis, Cell 74,    1089 (1993).-   27. K. V. Tarbell et al., J. Exp. Med. 196, 481 (2002).-   28. Q. Tang et al., J. Immunol. 171, 3348 (2003).-   29. T. Barthlott, G. Kassiotis, B. Stockinger, J. Exp. Med. 197, 451    (2003).

Example 3 Clinical Remission of Lupus Nephritis after Adoptive Transferof Expanded Treg Cells

Study size: Total number of subjects: 20; total number of sites 2

Study duration: 12-24 months

Target Population: Patients with lupus nephritis

Rationale: The importance of regulatory T lymphocytes (Treg) in thecontrol of autoimmunity is now well-established in a variety ofexperimental animal models (McHugh et al., The role of suppressor Tcells in regulation of immune responses. J Allergy Clin Immunol10:693-702, 2002). In addition, there are numerous studies suggestingthat Treg deficits may be an underlying cause of human autoimmunediseases. Most importantly, the emergence of CD4⁺CD25⁺ regulatory Tcells as an essential component of immune homeostasis provides apotential therapeutic opportunity for active immune regulation andlong-term tolerance induction. However, Tregs represent only a smallpercentage (<2%) of human CD4⁺ T cells, are reduced in number andfunction in autoimmune humans, and are generally considered to beproliferatively anergic. We have developed a potent method to expandTreg cells from humans. The cells, expanded up to 200-fold in less than3 weeks, express a classical Treg phenotype (CD4⁺, CD25⁺, CD62L^(hi),GITR⁺, and FoxP3⁺) and function to suppress T cell effectorproliferation and cytokine production.

The present study was designed to test the safety and efficacy ofCD4⁺CD25⁺ cells expanded from patients with lupus nephritis. Preliminarydata suggested that patients with systemic lupus erythematosus (SLE)demonstrate variability in Treg activity depending on the disease statuswith high Treg activity during remission and a decline at relapse(Crispin et al., Quantification of regulatory T cells in patients withsystemic lupus erythematosus. J Autoimmun 21:273-276, 2003). We soughtto determine whether we could identify, select and expand Treg frompatients at the time of remission, store those cells bycryopreservation, and reintroduce during relapse. The study was designedto test the safety of such autologous Treg therapy, and to determine theimpact of this therapy on disease course and immunologic parameters.

Study Design/Treatment Protocol: The study consists of two phases. Inthe first phase, we expand Treg from 5 patients with active lupusnephritis and 5 patients with a history of lupus nephritis in remission(off immunosuppressive therapy). This phase demonstrates the relativefeasibility of expanding Treg from patients with active and inactivedisease, and documents the functional capacity of the expanded cells.

The second phase is an open-label trial of Treg infusion in patientswith active lupus nephritis using two different design protocols. In thefirst protocol, we expand functional Treg from patients with activedisease, then these patients will immediately be the recipients of theirown expanded cells. Entry criteria include, in addition to active lupusnephritis, the presence of anti-dsDNA antibodies and hypocomplementemia.The entry criteria also include appropriate foundation therapy fornephritis (e.g., prednisone, mycophenolate mofetil, azathioprine, etc.).However, cyclophosphamide (CTX) therapy was an exclusion based onconcern that CTX may be more likely to interfere with the effects ofTreg. The primary endpoint is the safety of the autologous transfer,assessed by monitoring general clinical parameters and disease activity.The secondary endpoints (disease activity, serology, and mechanisticstudies) are measured at 30, 60, 90, 180, and 365 days post-treatment.

The second protocol is applied where sufficient Treg cannot be recoveredfrom patients with active lupus nephritis. Here, the cells are recoveredfrom patients during periods of disease remission, expanded ex vivo, andthen frozen in preparation for infusion at the time of relapse. Otherthan a larger patient pool and time period, the design of this trialparalleled the design described above.

The methods and materials parallel those described below for our Phase Istudy of adoptive cell transfer in diabetes patients. Our selectedFDA-approved anti-CD3/anti-CD28 coated beads and the specifiedmonoclonal antibodies are available from Xcyte Therapies and BectonDickinson, respectively. We have scaled the sorting and expansionprocedure to expand and cryopreserve about 109 Treg from individualpatients.

Primary Outcome: Safety

Secondary Outcomes: 1) Renal function, assessed by creatinine,proteinuria, and urinary sediment; 2) Lupus serology, assessed byanti-dsDNA antibodies and complement; 3) disease activity indices,assessed by SLEDAI and/or BILAG, and by patient global assessment; and4) Mechanistic studies.

Mechanistic Studies: 1) Measurement of anti-dsDNA and complement; 2)Assessment of the frequency of autoantibody-producing B cells; 3)Phenotypic analysis of circulating lymphocytes to detect Treg phenotype;4) Assessment of Treg activity; and 5) ELISPOT for Th1 and Tregcytokines.

Interpretation. This study demonstrates that Treg cells derived frompatients with lupus nephritis have no untoward effects on health ordisease progression in the patients, and that adoptive transfer therapyimproves renal function in these patients.

Example 4 Clinical Remission of Diabetes Mellitus after AdoptiveTransfer of Expanded Treg Cells

This trial demonstrates clinical remission of diabetes mellitus afteradoptive transfer of expanded Treg cells. Our study lymphocytecollection and infusion protocols were adapted from Rapoport, et al.:Molecular remission of CML after autotransplantation followed byadoptive transfer of costimulated autologous T cells. Bone MarrowTransplant (Epub ahead of print, Oct. 27, 2004).

Patient eligibility and enrollment. We required that prior written andinformed consent be obtained from all patients, in accordance with theInstitutional Review Board guidelines. Patients are required to havediabetes mellitus based on characteristic clinical and laboratoryfeatures. Adequate renal, cardiac, pulmonary, and hepatic functions arerequired, and patients may not have active infections or HIVseropositivity.

Steady-state lymphocyte collection. Patients first undergo asteady-state leukapheresis using an automated cell separator (CobeSpectra cell collector or equivalent). Approximately 20-30 l of blood isprocessed through a large bore catheter, to obtain about 1.5×10⁸mononuclear cells per kg body weight. These cells are cryopreserved forlater expansion in our anti-CD3/anti-CD28 culture system.

Ex vivo costimulation and expansion of T-lymphocytes. T cells arecultured, as specified, in an FDA-approved investigational new drugapplication. Around day 0 of the autotransplantation phase, thecryopreserved mononuclear cells are thawed and washed three times in PBSwith 1% human serum albumin. If not performed at the time of initialcryopreservation, the mononuclear cells are monocyte-depleted usingmagnetic beads in a closed system. The cells are then seeded intogas-permeable flasks (Baxter Oncology, Deerfield, Ill., USA) containingX-VIVO supplemented with 5% pooled AB serum. Paramagnetic beads withimmobilized anti-CD3 (OKT3) and anti-CD28 (9.3) monoclonal antibodiesare added at a 2:1 bead:CD3+ cell ratio, and the IL-2 (1000 IU/ml)supplemented cultures maintained for up to 14 days prior to harvest andpreparation for infusion (supra). The cells are counted daily and freshmedium supplemented with IL-2 at 1000 IU/ml is added to maintain thecells at a density of 0.75-2×10⁶/ml. After completion of cell culture,the magnetic beads are removed using a Baxter Fenwal Maxsep magneticcell separation device. After removal of the beads, the cells arewashed, concentrated and resuspended in 100-250 ml of Plasmalyte Acontaining 1% human serum albumin, using the Baxter Fenwal HarvesterSystem.

Reinfusion of ex vivo expanded T cells. Before release, all theharvested products are required to meet the criteria for cell viability(70%), sterility (negative cultures for bacteria and fungi, negativeendotoxin assay), and bead contamination (<100 beads/3×10⁶ cells). Theharvested cells are transported by courier from the cell productionfacility to the patient and infused on the same day. The cells areinfused over 20-60 min without a leukocyte filter. Patients may beroutinely premedicated with acetaminophen and diphenhydramine.

Example 5 Clinical Remission of Diabetes Mellitus after AdoptiveTransfer of Expanded Treg Cells

This trial demonstrates clinical remission of diabetes mellitus afteradoptive transfer of expanded Treg cells. Our study lymphocytecollection and infusion protocols were adapted from Rapoport, et al.:Molecular remission of CML after autotransplantation followed byadoptive transfer of costimulated autologous T cells. Bone MarrowTransplant (Epub ahead of print, Oct. 27, 2004).

Patient eligibility and enrollment. We required that prior written andinformed consent be obtained from all patients, in accordance with theInstitutional Review Board guidelines. Patients are required to havediabetes mellitus based on characteristic clinical and laboratoryfeatures. Adequate renal, cardiac, pulmonary, and hepatic functions arerequired, and patients may not have active infections or HIVseropositivity.

Steady-state lymphocyte collection. Patients first undergo asteady-state leukapheresis using an automated cell separator (CobeSpectra cell collector or equivalent). Approximately 20-30 l of blood isprocessed through a large bore catheter, to obtain about 1.5×10⁸mononuclear cells per kg body weight. These cells are cryopreserved forlater expansion in our MHC class II molecule/peptidecomplex—costimulatory agent culture system.

Ex vivo costimulation and expansion of T-lymphocytes. T cells arecultured, as specified, in an FDA-approved investigational new drugapplication. Around day 0 of the autotransplantation phase, thecryopreserved mononuclear cells are thawed and washed three times in PBSwith 1% human serum albumin. If not performed at the time of initialcryopreservation, the mononuclear cells are monocyte-depleted usingmagnetic beads in a closed system. The cells are then seeded intogas-permeable flasks (Baxter Oncology, Deerfield, Ill., USA) containingX-VIVO supplemented with 5% pooled AB serum. Paramagnetic beads withimmobilized MHC Complex type II DQ0602/insulinB peptide (aa5-15) andanti-CD28 (9.3) monoclonal antibodies are added at a 2:1 bead:CD3+ cellratio, and the IL-2 (1000 IU/ml) supplemented cultures maintained for upto 14 days prior to harvest and preparation for infusion (supra). Thecells are counted daily and fresh medium supplemented with IL-2 at 1000IU/ml is added to maintain the cells at a density of 0.75-2×10⁶/ml.After completion of cell culture, the magnetic beads are removed using aBaxter Fenwal Maxsep magnetic cell separation device. After removal ofthe beads, the cells are washed, concentrated and resuspended in 100-250ml of Plasmalyte A containing 1% human serum albumin, using the BaxterFenwal Harvester System.

Reinfusion of ex vivo expanded T cells. Before release, all theharvested products are required to meet the criteria for cell viability(70%), sterility (negative cultures for bacteria and fungi, negativeendotoxin assay), and bead contamination (<100 beads/3×10⁶ cells). Theharvested cells are transported by courier from the cell productionfacility to the patient and infused on the same day. The cells areinfused over 20-60 min without a leukocyte filter. Patients may beroutinely premedicated with acetaminophen and diphenhydramine.

The foregoing descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation. Unlesscontraindicated or noted otherwise, in these descriptions and throughoutthis specification, the terms “a” and “an” mean one or more, the term“or” means and/or. All publications and patent applications cited inthis specification and all publications cited therein are hereinincorporated by reference as if each individual publication werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. TABLE A Autoimmune disease AutoantigenMHC class II molecule/peptide(s) bound Lupus erythematosus giantingolgin-245/p230 golgin-160/GCP170 golgin-95/GM130 golgin-97 golgin-67transferrin 119 - VVKKGTDFQLNQLEGKK (SEQ ID NO: 1) 119 -VVKKGTDFQLNQLGKK (SEQ ID NO: 2) [see Freed et al., J. Immunol. (2000)164: 4697-4705 (ref 1)] A_(β) ^(k) (37-51 major; YVRFDSDVGEYRAVTE (SEQID 37-52 minor) NO: 3) (ref 1) Lysozyme c (48-63) GDQSTDYGIFQINSRY (SEQID NO: 4) (ref 1) nucleoporin NUP155 RQVRFYSGVIEL (SEQ ID NO: 5) (ref(120-) 1) Saposin D (37-) LPDPYQKQCDDFVAE (SEQ ID NO: 6) (ref 1) 26Sproteasome IFLDDPQAVSDVL (SEQ ID NO: 7) p112 (224-) (ref 1) 14-3-3protein β, δ, KTAFDEAIAELD (SEQ ID NO: 8) ( ref ζ, θ, or τ (95-) 1)A^(k) _(β)(143-) STQLIRNGDWTFQVLVMLEM (SEQ (110-) ID NO: 9)HHNTLVCSVTDFYPAKIKVR (SEQ ID NO: 10) (ref 1) Ig γ 1-chain (141-)SMVTLGCLVKGYFPEPVTVT (SEQ ID NO. 11) (ref 1) Thrombocytopenic GPIIb/IIIaHLA-DR purpura (Kuwana et al., J Clin Invest. 1998 Oct 1; 102(7):1393-402) platelet integrin Goodpasture's human glomerular syndromebasement membrane Graves disease thyroglobulin thyroperoxidasesodium-iodide symporter TSH receptor Type I diabetes Insulin, proinsulinDQ0601/insulin B aa5-15 mellitus aa1-15: FVNQHLCGSHLVEAL (SEQ ID NO: 12)(see Ettinger and Kwok, J Immunol. 1998 Mar 1; 160(5): 2365-73) HLA-DR3glutamic acid HLA-DR4 (DRB1*0401)/271-285 decarboxylase(PRLIAFTSEHSHFSL) (SEQ ID (GAD65) NO: 13) 116-130 (NILLQYVVKSFDRST) (SEQID NO: 14); HLA-DR4 (DRA1*0101)/356- 370 (KYKIWMHVDAAWGGG) (SEQ ID NO:15), 376-390 (KHKWKLNGVERANSV) (SEQ ID NO: 16), 481-495(LYNIIKNREGYEMVF) (SEQ ID NO: 17), 511-525 (PSLRVLEDNEERMSR) (SEQ ID NO:18), 546-560 (SYQPLGDKVNFFRMV) (SEQ ID NO: 19), 556-570(FFRMVISNPAATHQD) (SEQ ID NO: 20), and 566-580 (ATHQDIDFLIEEIER) (SEQ IDNO: 21); HLA-DQ8/206-220 (TYEIAPVFVLLEYVT) (SEQ ID NO: 22) (see Peng, Y.Chin Med J 2001; 114(10): 229-242) tyrosine phosphatase IA-2 tyrosinephosphatase 2b IGRP Human protein: Q9UN79-SOX-13 protein (Type 1diabetes autoantigen ICA12) (Islet cell antigen 12). ICA69 Myastheniagravis Gravin muscle nicotinic 121-136 (PAIFKSYCEIIVTHFP) (SEQacetylcholine ID NO: 23) receptor (AChR) 129-145 (EIIVTHFPFDEQNCSMK)(SEQ ID NO: 24) [see J Immunol 159(3): 1570-7] p195-212(DTPYLDITYHFVMQRLPL) (SEQ ID NO: 25) [see Scand J Immun. 44(5): 512-21]Pemphigus vulgaris desmoglein 1, desmoglein 3, Human desmocollin 1(Dsc1) bullous pemphigoid BP180 Autoimmune Formiminotransferasehepatitis cyclodeaminase Autoimmune atrophic parietal cell H, K- corpusgastritis adenosine triphosphatase (ATPase) Addison's disease CYP21CYP17 CYP11A1 Rheumatoid arthritis endoplasmic reticulum molecularchaperone immunoglobulin binding protein (BiP) human cartilage HLA-DR4(DRB1*0401)/aa259-271 glycoprotein-39 (PTFGRSFTLASSE) (SEQ ID NO: 26)(YKL40) (see Vos et al, Rheumatology (2000) 39: 1326-1331) type IIcollagen glucose-6-phosphate isomerase Multiple sclerosis alphaβ-Crystallin DRB1*1501 myelin HLA-DR4 (DRB1*0401)/97-108 oligodendrocyte(TCFFRDHSYQEE) (SEQ ID NO: 27) glycoprotein (MOG) (see Forsthuber et al,J Immunol. 2001 Dec 15; 167(12): 7119-25) Myelin basic protein 111-119(SLSRFSWGA) (SEQ ID (MBP) NO: 28) and 87-95 (VVHFFKNIV) (SEQ ID NO: 29presented in HLA- A2 and HLA-A24 [see JI, 172(8): 5120-7] X2MBPPsoriasis Cytokeratin 17 cutaneous lymphocyte antigen (CLA) Autoimmuneanion channel 861-874 (CLAVLWVVKSTPAS) (SEQ hemolytic anemia proteinband 3 ID NO: 30 [see Blood 15; 102(10): 3800-6] Uveitis S-antigen341-354 (FLGELTSSEVATEV)(SEQ ID NO: 31) [see Int. Immun., 15(8):927-935] interphotoreceptor retinoid-binding protein (IRBP) HLA-B(B27PD)125-138 ALNEDLSSQTAADT (SEQ ID NO: 32) [see Int. Immun., 15(8): 927-935]

1. A method of modulating an autoimmune response in a subject, saidmethod comprising: obtaining a population of subject-compatible cells;producing a predetermined autoantigen-specific regulatory T cellenriched composition from said population of cells; and introducing saidcomposition into said subject to modulate said autoimmune response insaid subject.
 2. The method according to claim 1, wherein saidpopulation of cells is obtained from said subject.
 3. The methodaccording to claim 1, wherein said population of cells is obtained froma donor distinct from said subject.
 4. The method according to claim 1,wherein said population of cells is harvested from peripheral blood. 5.The method according to claim 1, wherein said producing step comprisesexpanding said antigen-specific regulatory T cells.
 6. The methodaccording to claim 5, wherein said expanding is achieved by contactingsaid population of cells with an autoantigen-specific regulatory T cellstimulatory composition.
 7. The method according to claim 5, whereinregulatory T cells are enriched from said population of cells prior tosaid expanding step.
 8. The method according to claim 5, whereinregulatory T cells are enriched from said population after saidexpanding step.
 9. The method according to claim 6, wherein saidstimulatory composition comprises an MHC class II/autoantigenic peptidecomplex.
 10. The method according to claim 6, wherein said stimulatorycomposition comprises a costimulatory agent.
 11. The method according toclaim 10, wherein said costimulatory agent is an agonist antibody. 12.The method according to claim 11, wherein said agonist antibody binds toCD28.
 13. The method according to claim 6, wherein said stimulatorycomposition comprises a second regulatory T cell stimulatory agent. 14.The method according to claim 13, wherein said second stimulating agentis a cytokine.
 15. The method according to claim 14, wherein saidcytokine is an interleukin.
 16. The method according to claim 15,wherein said interleukin is interleukin-2.
 17. The method according toclaim 6, wherein said stimulatory composition is immobilized on asubstrate.
 18. The method according to claim 17, wherein said substrateis a cell.
 19. The method according to claim 17, wherein said substrateis a bead.
 20. The method according to claim 1, wherein said producingstep comprises enriching said autoantigen-specific regulatory T cellsfrom said obtained population of cells.
 21. The method according toclaim 1, wherein said modulating comprises inhibiting. 22-40. (canceled)